Journal: Science Advances

Article Title: MYC-bound enhancer RNAs in cis regulate gene transcription and tumorigenesis

doi: 10.1126/sciadv.aeb5147

Figure Lengend Snippet: ( A ) Genomic tracks of MYC ChIP-seq, MYC eCLIP-seq, and GRO-seq signals at the distal and proximal regions of the GREB1 promoter in MCF-7 cells. POLR2A-mediated chromatin interactions by ChIA-PET were shown at the bottom. ( B ) Representative two-color super-resolution (STORM) images depicting the spatial relationship between MERG1 eRNA transcripts and MYC proteins within the nucleus of MCF-7 cells. Scale bars, 1 μm (main image) and 200 nm (insets). ( C ) Correlation of the MERG1 level with GREB1 and E2F6 levels by qRT-PCR in breast cancer cell lines. ( D ) qRT-PCR for MERG1 , GREB1 , E2F6 , and ROCK2 levels and Western blot for GREB1 in BT474 cells after 2 days of transfection with ASOs targeting MERG1 (ASO-MERG1). A scrambled ASO served as control (ASO-Ctrl). ( E ) qRT-PCR and Western blot for EXOSC3 level in MCF-7 cells stably expressing shRNA targeting EXOSC3 (shEXOSC3) or scramble control (shCtrl). ( F ) Volcano plot showing the differential expression of FANTOM5 eRNAs in MCF-7 cells stably expressing shEXOSC3-B in (E). eRNAs were considered differentially regulated if the log 2 (fold change) > 1 and P < 0.05. MERG1 is highlighted in orange. ( G ) qRT-PCR for MERG1 , GREB1 , E2F6 , and ROCK2 levels in MCF-7 cells in (E) (representative data from three independent experiments). ( H ) Correlation of expression or enhancer chromatin accessibility of MERG1 ( x axis) with GREB1 , E2F6 , and ROCK2 in breast cancer using CCLE RNA-seq, TCGA ATAC-seq, and TCGA RNA-seq. The peaks used from TCGA ATAC-seq were MERG1 , LGG_8176; GREB1 , UCEC_12596; E2F6 , BLCA_11756; and ROCK2 , COAD_12529. The plot and error bar indicate the mean and SD of technical replicates ( n = 3) in (D), (E), and (G).

Article Snippet: Human breast cancer cell lines, including BT-20, BT474, HCC1937, Hs578T, MCF-7, MDA-MB-231 (MB231), MDA-MB-361 (MB361), MDA-MB-468 (MB468), T-47D, and ZR-75-1, and human mammary epithelial cell line MCF10A, human lung cancer cell line A549, and human embryonic kidney (HEK) 293T cell line were directly obtained from the American Type Culture Collection (ATCC).

Techniques: ChIP-sequencing, ChIA Pet Assay, Quantitative RT-PCR, Western Blot, Transfection, Control, Stable Transfection, Expressing, shRNA, Quantitative Proteomics, RNA Sequencing

Journal: Science Advances

Article Title: MYC-bound enhancer RNAs in cis regulate gene transcription and tumorigenesis

doi: 10.1126/sciadv.aeb5147

Figure Lengend Snippet: ( A ) qRT-PCR for RNA distribution in whole cell lysate (WCL), cytoplasmic (Cyto), soluble nuclear (Nuc), and chromatin-associated (Chr) fractions of MCF-7 cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), SNRP70, and Histone 3 served as fraction markers. ( B ) Representative wide-field IF staining of MYC and RNA FISH of MERG1 and nascent GREB1 transcripts in a single MCF-7 cell. Scale bars, 2 μm or 200 nm. DAPI, 4′,6-diamidino-2-phenylindole. ( C ) Schematic representing the GAL4-λN- BoxB tethering assay of MERG1 RNA regulating the GREB1 promoter to mimic promoter-enhancer interaction. BoxB RNA motif binds the λN peptide fused to the DNA binding domain of GAL4 (GAL4-λN), which is tethered to five GAL4 binding sites (5×UAS) downstream of the MERG1 cognate enhancer ( MERG1en , brown). MERG1en inserted ~2.8 kb upstream of the GREB1 promoter ( GREB1p , blue) of the luciferase (Luc) reporter. ( D and E ) MERG1 rescues the loss of luciferase activity of MERG1en -Δ MERG1 reporter. The schematic in (D) represents the GAL4-λN-BoxB tethering assay. BoxB , BoxB - GFP , or BoxB - MERG1 RNA is recruited to the upstream of the reporter. Reporter carries a control DNA fragment (gray), the MERG1en (brown), or the MERG1en - ΔMERG1 (yellow). BoxB and BoxB - GFP served as controls. MERG1en - ΔMERG1 lacks the DNA sequence that can be transcribed into MERG1 . ( F ) MERG1 augments MERG1en reporter activity in a dose-dependent manner. ( G ) ASO-mediated MERG1 knockdown attenuates MERG1en reporter activity. ( H and I ) ChIP-qPCR for MYC (H), and BRD4, p300, and H3K27ac (I) occupancy at the MERG1en and GREB1p in BT474 cells treated with ASO-MERG1. The plot and error bar indicate the mean and SD of biological replicates ( n = 3) in (E) to (G) and technical replicates ( n = 3) in (A), (H), and (I). P values were determined by unpaired two-sided t test in (E) to (G). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Human breast cancer cell lines, including BT-20, BT474, HCC1937, Hs578T, MCF-7, MDA-MB-231 (MB231), MDA-MB-361 (MB361), MDA-MB-468 (MB468), T-47D, and ZR-75-1, and human mammary epithelial cell line MCF10A, human lung cancer cell line A549, and human embryonic kidney (HEK) 293T cell line were directly obtained from the American Type Culture Collection (ATCC).

Techniques: Quantitative RT-PCR, Staining, Binding Assay, Luciferase, Activity Assay, Control, Sequencing, Knockdown, ChIP-qPCR

Journal: Science Advances

Article Title: MYC-bound enhancer RNAs in cis regulate gene transcription and tumorigenesis

doi: 10.1126/sciadv.aeb5147

Figure Lengend Snippet: ( A ) Comparison of MERG1 levels in TCGA BRCA tumor ( n = 1095) and normal ( n = 113) samples. P value was determined by the Mann-Whitney U test. ( B ) MTT assay for cell growth after 2 days in BT474 and MCF-7 cells transfected with ASO-MERG1. ( C to E ) qRT-PCR for MERG1 levels (C), flow cytometry for cell cycle phase distribution (D), and MTT assay for cell growth (E) of BT474 and MCF-7 cells stably expressing shMERG1. ( F ) Growth curve (left) and representative tumor size (right) of the xenograft mouse model bearing BT474 (top) and MCF-7 (bottom) cells stably expressing shMERG1 or shCtrl. ( G ) Timeline for the BT474-xenograft in vivo mouse model treated with the ASO-MERG1 or ASO-Ctrl loaded into PMBOP NPs (PMBOP/ASO), and a 1.5 mg/kg dosage of ASOs was used. Arrows indicate different events: black, cell inoculation; red, phosphate-buffered saline (PBS) or PMBOP/ASO injection; green, tumor collection. ( H ) Growth curve (left) and representative tumor size (right) of BT474-xenograft in vivo mouse model treated with PMBOP/ASO NPs. PBS solution and ASO-Ctrl served as controls. ( I ) A model of the cis-regulatory role of MERG1 on GREB1 transcription in an MYC-dependent manner. The plot and error bar indicate the mean and SD of biological replicates [ n = 4 in (B) and (E), n = 6 in (F), and n = 5 in (H)], and technical replicates [ n = 3 in (C) and (D)]. P values were determined by unpaired two-sided t test in (B), (E), (F), and (H). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Human breast cancer cell lines, including BT-20, BT474, HCC1937, Hs578T, MCF-7, MDA-MB-231 (MB231), MDA-MB-361 (MB361), MDA-MB-468 (MB468), T-47D, and ZR-75-1, and human mammary epithelial cell line MCF10A, human lung cancer cell line A549, and human embryonic kidney (HEK) 293T cell line were directly obtained from the American Type Culture Collection (ATCC).

Techniques: Comparison, MANN-WHITNEY, MTT Assay, Transfection, Quantitative RT-PCR, Flow Cytometry, Stable Transfection, Expressing, In Vivo, Saline, Injection

Journal: International Journal of Molecular Medicine

Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

doi: 10.3892/ijmm.2026.5751

Figure Lengend Snippet: EBA impairs cancer stem cell-like properties. (A) BT474 and SKBR3 cells were treated with EBA for 48 h, and ALDH1 activity was assessed by flow cytometry using the Aldefluor assay. DEAB was used to define the baseline of Aldefluor-positive fluorescence. (B) BT474 cells (5x10 4 cells/ml) were plated in ultra-low attachment dishes and cultured in the presence or absence of EBA for 5 days. The number and volume of mammospheres were measured by microscopy. (C) Overall survival of patients with breast cancer stratified by the co-expression of ALDH1A1 and CD44. (D) Spearman correlation analysis of ALDH1A1 and CD44 mRNA levels in patients with HER2-positive breast cancer from The Cancer Genome Atlas cohort (n=76). Kaplan-Meier survival analyses of patients with HER2-overexpressing breast cancer stratified by (E) ALDH1A1 and (F) CD44 expression. Patients were divided into high- and low-expression groups based on the median gene expression. Statistical significance was determined using the log-rank test. (G) JIMT-1 cells were treated with EBA (3 μ M) for 48 h and the CD44 high /CD24 low cell populations were identified by flow cytometry. (H) JIMT-1 cells (1.5x10 4 cells/ml) were cultured under serum-free suspension conditions in the presence of EBA (3 μ M) for 8 days. Mammosphere number and volumes were quantified. ** P<0.01 and **** P<0.0001 vs. vehicle-treated control (0 μ M EBA). EBA, ebastine; ALDH, aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; CTL, control; ISO, isotype.

Article Snippet: The human breast cancer cell lines SKBR3, BT474, MDA-MB-453 (American Type Culture Collection) and JIMT-1 (Leibnitz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH) were cultured in DMEM, MEM or RPMI-1640 (all Sigma-Aldrich; Merck KGaA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Activity Assay, Flow Cytometry, Fluorescence, Cell Culture, Microscopy, Expressing, Gene Expression, Suspension, Control

Journal: PLoS ONE

Article Title: Chronic Morphine Treatment Attenuates Cell Growth of Human BT474 Breast Cancer Cells by Rearrangement of the ErbB Signalling Network

doi: 10.1371/journal.pone.0053510

Figure Lengend Snippet: ( A ) Detection of µ-opioid receptors by RT-PCR. Reactions contained 10 ng of cDNA from BT474 cells or from the corresponding cloned receptors (Cn). Only a 293 bp fragment of the µ-opioid receptor is present (arrow). Control PCRs with primers for GAPDH or water instead of cDNA demonstrated the integrity of cDNA and the specificity of the RT-PCR reaction. Ladder: 100 bp. ( B ) Determination of G proteins in membranes from BT474 cells by Western blot using subtype-specific antibodies. BT474 cells contain inhibitory G i α2 and G i α3 as well as stimulatory G s α subunits. They also contain G q/11 α, G 12 α, G 13 α, G 14 α and Gβ subunits. Membranes of MCF-7 cells served as control. ( C ) Identification of adenylyl cyclase (AC) isoforms in BT474 cells by RT-PCR. Reactions contained 10 ng of cDNA and primer pairs for all mammalian AC types. The following fragments were amplified (arrows): AC type 1 (143 bp), 2 (323 bp), 3 (259 bp), 4 (368 bp), 5 (312 bp), and 9 (325 bp). The quality of cDNA was verified by amplification of GAPDH, the specificity by using water instead of cDNA. Ladder: 100 bp. ( D ) Regulation of intracellular cAMP production by opioids. Control and chronically Morphine treated BT474 cells were stimulated for 15 min with Forskolin (10 µM). µ-Opioid receptor-mediated inhibition of cAMP production was assessed in the presence of Morphine (10 µM). Receptor specificity was determined by co-incubation with Naloxone (100 µM). The data shown are from 3 experiments. ***; significantly different at p<.001. n.s.; not quite significant.

Article Snippet: BT474 cells were purchased from Cell Lines Service Inc. and grown in RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf serum (FCS), 100 IU/ml Penicillin, 0.1 mg/ml Streptomycin, 0.2% Enrofloxacin and 0.02 mg/ml Insulin at 37°C in a humidified atmosphere of 5% CO2 in air.

Techniques: Reverse Transcription Polymerase Chain Reaction, Clone Assay, Western Blot, Amplification, Inhibition, Incubation

Journal: PLoS ONE

Article Title: Chronic Morphine Treatment Attenuates Cell Growth of Human BT474 Breast Cancer Cells by Rearrangement of the ErbB Signalling Network

doi: 10.1371/journal.pone.0053510

Figure Lengend Snippet: ( A ) BT474 cells were cultured for 5 d in the presence or absence of Morphine (10 µM), Naloxone (100 µM), and Heregulin (40 ng/ml), before cell growth was determined by crystal violet staining. Top: Photograph of tissue culture wells from a representative experiment before solubilisation of the dye. Bottom: Data of n = 6 independent experiments normalized to controls. Note that co-incubation of the cells with Morphine significantly attenuates Heregulin-stimulated cell growth (**, p<.005). ( B ) BT474 cell migration was assessed by the scratch assay done in cells grown for 5 d in the absence (control) or presence of Heregulin (40 ng/ml), Morphine (10 µM) and Naloxone (100 µM) as indicated. After scratching, cells were kept for another 24 h in the presence of the above ligands, before images were acquired using an Olympus BH-2 microscope (40× magnification). The figures shown are representative for 3 independent experiments yielding qualitatively similar results.

Article Snippet: BT474 cells were purchased from Cell Lines Service Inc. and grown in RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf serum (FCS), 100 IU/ml Penicillin, 0.1 mg/ml Streptomycin, 0.2% Enrofloxacin and 0.02 mg/ml Insulin at 37°C in a humidified atmosphere of 5% CO2 in air.

Techniques: Cell Culture, Staining, Incubation, Migration, Wound Healing Assay, Microscopy

Journal: PLoS ONE

Article Title: Chronic Morphine Treatment Attenuates Cell Growth of Human BT474 Breast Cancer Cells by Rearrangement of the ErbB Signalling Network

doi: 10.1371/journal.pone.0053510

Figure Lengend Snippet: ( A ) Determination of Akt activation in control and chronically Morphine (10 µM; 5d)-treated cells. Cells were incubated for 5 min at 37°C in the presence or absence of Morphine (10 µM), Naloxone (100 µM) and Heregulin (40 ng/ml), before Akt phosphorylation was determined by Western blot using a phosphor-specific antibody. The overall amount of Akt was determined by a phosphorylation-insensitive antibody. Insets show representative Western blots of the corresponding proteins (60 kDa) and β-tubulin (loading control). Immunoreactive bands were quantified and normalized to Heregulin-stimulated values in control cells, which was set to 100%. The data shown are from n = 4 independent experiments. ( B ) Comparison of basal and Heregulin (40 ng/ml)-stimulated Akt activation in control and Morphine (10 µM; 5d)-treated cells. Samples were run on the same gel and stained for phospho-Akt, total Akt and β-tubulin as loading control. Note that chronic morphine treatment increases basal and Heregulin (40 ng/ml)-stimulated levels of Akt phosphorylation. ( C ) Determination of PARP cleavage in BT474 cells. Cells were cultured in the absence (control) or presence of Morphine (10 µM; 5 d), before cells were washed and grown for an additional 6 h in serum-free Medium either in the absence or presence of Heregulin (40 ng/ml) Morphine (10 µM) and Naloxone (100 µM) as indicated. Samples were analysed by Western blot using an antibody recognizing full length (116 kDa) and cleaved (89 kDa) PARP. The same samples were blotted for β-tubulin (loading control). ( D ) Determination of apoptosis by Annexin V/propidium iodide staining. BT474 cells were cultured on coverslips for 5 d in the presence or absence of Morphine (10 µM), Naloxone (100 µM) and Heregulin (40 ng/ml) alone or in combination as indicated. Cells were sequentially stained with Annexin-FITC (green), propidium iodide (red), fixed and analysed by confocal microscopy. Bar: 20 µm.

Article Snippet: BT474 cells were purchased from Cell Lines Service Inc. and grown in RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf serum (FCS), 100 IU/ml Penicillin, 0.1 mg/ml Streptomycin, 0.2% Enrofloxacin and 0.02 mg/ml Insulin at 37°C in a humidified atmosphere of 5% CO2 in air.

Techniques: Activation Assay, Incubation, Western Blot, Staining, Cell Culture, Confocal Microscopy

Journal: PLoS ONE

Article Title: Chronic Morphine Treatment Attenuates Cell Growth of Human BT474 Breast Cancer Cells by Rearrangement of the ErbB Signalling Network

doi: 10.1371/journal.pone.0053510

Figure Lengend Snippet: ( A ) Effect of protein kinase blockers on basal and Heregulin-stimulated ERK1/2 and Akt phosphorylation. BT474 cells were cultured for 5 d in the absence (control) or presence of Morphine (10 µM), before the impact of ErbB1 (AG1478), ErbB2 (AG825), PI3K (Wortmannin; Wort.) and metalloproteinases (EGCG) on basal and Heregulin (40 ng/ml)-stimulated ERK1/2 and Akt signalling was determined by Western blot using phospho-specific antibodies. Controls were kept in the absence of protein inhibitors. To verify equal protein loading, controls were also stained with overall reactive anti-ERK1/2 and anti-Akt antibodies. ( B ) Involvement of ErbB2 on basal and EGF-stimulated ERK1/2 and Akt signalling. Controls and cells chronically exposed to Morphine (10 µM; 5d) were incubated with or without AG825 (50 µM; 30 min), before ERK1/2 and Akt phosphorylation was determined for 5 min in the absence or presence of EGF (100 ng/ml). Equal protein loading was verified by staining controls with overall reactive anti-ERK1/2 and anti-Akt antibodies. ( C ) Regulation of ErbB receptor abundance by chronic Morphine treatment in BT474 cells. Cells were cultured for 5 d in the presence or absence of Morphine (10 µM) and Naloxone (100 µM) as indicated and overall ErbB receptor levels were analysed by Western blot using specific antibodies for ErbB1 (175 kDa), ErbB2 (185 kDa), ErbB3 (185 kDa) and ErbB4 (170 kDa). Equal protein loading was verified by incubation of the blots with an antibody against β-tubulin. ( D ) Alteration of ErbB1 containing receptor dimers by chronic Morphine. Controls and chronically Morphine (10 µM; 5 d)-treated BT474 cells were stimulated for 5 min with or without Heregulin (40 ng/ml) to form receptor dimers. Proteins were cross-linked and ErbB1 containing dimers were immunoprecipitated using an anti-ErbB1 antibody. Individual ErbB receptors were determined in whole cell solubilisates (S) and immunoprecipitates (IP) by Western blot using type-specific antibodies. Equal protein load was verified by determination of IgG heavy chains ( F c ).

Article Snippet: BT474 cells were purchased from Cell Lines Service Inc. and grown in RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf serum (FCS), 100 IU/ml Penicillin, 0.1 mg/ml Streptomycin, 0.2% Enrofloxacin and 0.02 mg/ml Insulin at 37°C in a humidified atmosphere of 5% CO2 in air.

Techniques: Cell Culture, Western Blot, Staining, Incubation, Immunoprecipitation

Journal: PLoS ONE

Article Title: Chronic Morphine Treatment Attenuates Cell Growth of Human BT474 Breast Cancer Cells by Rearrangement of the ErbB Signalling Network

doi: 10.1371/journal.pone.0053510

Figure Lengend Snippet: The scheme depicts the differences in Heregulin-stimulated ErbB signalling in control and chronically Morphine-treated BT474 cells. In control cells, stimulation with Heregulin leads to activation of ERK1/2 and Akt signalling via ErbB1/ErbB3 heterodimers. While AG1478 blocks both Heregulin-stimulated ERK1/2 and Akt signalling, inhibition of PI3K by Wortmannin specifically prevents Akt signalling. Because ErbB3 phosphorylation and PI3K activation is dependent on the presence of a dimerized ErbB member with catalytic activity, these results indicate that Heregulin-stimulated ERK1/2 signalling is predominantly mediated through ErbB1. The failure of Wortmannin to affect ERK1/2 activation in control cells further implicates that Heregulin stimulates Akt signalling via ErbB3. Chronic Morphine treatment alters mitogenic signalling by rearrangement of ErbB heterodimers. Whereas Heregulin still stimulates Akt signalling via ErbB1/ErbB3, ERK1/2 signalling is now accomplished by ErbB1/2 heterodimers. These are activated indirectly by an EGF-like ligand liberated from Heregulin in a PI3K (Wortmannin)- and metalloproteinase (EGCG)-dependent manner.

Article Snippet: BT474 cells were purchased from Cell Lines Service Inc. and grown in RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf serum (FCS), 100 IU/ml Penicillin, 0.1 mg/ml Streptomycin, 0.2% Enrofloxacin and 0.02 mg/ml Insulin at 37°C in a humidified atmosphere of 5% CO2 in air.

Techniques: Activation Assay, Inhibition, Activity Assay